Acute Promyelocytic Leukemia (APL) is associated with reciprocal translocations which involve the RARa locus on chromosome 17 which variably translocates and fuses to the PML, PLZF, NPM or NuMA genes (for brevity referred to as X genes/proteins). In all cases, due to the reciprocity of the translocation, two fusion genes are generated encoding X-RARa and RARa-X fusion proteins. We hypothesize that X-RARa and RARa-X act to interfere with the normal transcriptional function of RARa and the biological function of X proteins, and that X proteins thus play a key role in oncogenesis. We propose to test the above hypothesis and study the function of the X genes in both APL and human cancer pathogenesis with the following Specific Aims: 1) To define the molecular basis underlying PML tumor suppressive activity and the aberrant mechanism(s) that causes its loss in human cancer. We determined in knock-out (KO) mice and cells that nuclear isoforms of PML exerts key tumor suppressive functions. More recently, we discovered that PML also acts in the cytosol through isoforms devoid of a nuclear localization signal (cytoplasmic PML: cPML). However, to date no biochemical discrete function has been attributed to PML. We also demonstrated that PML is frequently lost in multiple prevalent human cancers. We propose to study whether PML displays a specific intrinsic enzymatic activity;to determine the mechanisms of PML loss in human cancer;and to further investigate the biological function of cPML. 2) To determine the role of PLZF and its homologue PLZP in hemopoietic stem cell (HSC), leukemia stem cell (LSC) biology and leukemogenesis. We showed in KO mice that Plzf exerts important developmental and tumor/growth suppressive functions. Recently, we determined that Plzf is critical for the quiescence and maintenance of the stem cell compartment. We will test whether Plzf is essential for the self-renewal capabilities of LSCs of acute myeloid leukemia (AML) subtypes and for the generation of quiescent long term (LT) - LSCs that are key to disease relapse. We will study hematopoiesis and HSC compartment biology in Plzf/Plzp double KO mutants, which we have recently generated. Lastly, we-will assess both in vitro and in vivo the functional relevance of an emerging negative cross talk between Plzf and Pokemon. 3) To determine the role of NPM in tumor suppression, myelodysplastic syndrome (MDS) and leukemia pathogenesis. We have generated a Npm hypomorphic/KO allelic series in the mouse and found that Npm inactivation results in cancer susceptibility. Importantly, Npm+l- mice spontaneously develop a MDS-like syndrome. We will determine the mechanisms by which Npm exerts its tumor suppressive function focusing oh the cross talk between Npm and the Arf/p53 pathway. We will study the role of NPM in MDS pathogenesis in Npm+l- cells/mice, in Npm conditional mutants, in mice concomitantly lacking Npm and other candidate genes, on 5q and in human MDS samples. 4) To define in KO mice the role of NuMA in ontogenesis, hemopoiesis and oncogenesis. We will characterize Numa complete and conditional KO mice to investigate its role in development and hemopoiesis. Numa-/- primary cells (e.g. mouse embryonic fibroblasts) and Numa mouse mutants will be employed to study the role of this gene in tumorigenesis.